Description: Aminopeptidases (APs) are metalloproteases that cleave amino-terminal (N-terminal) amino acids during protein synthesis (1, 2) These enzymes are characterized in part by their post-translational removal of leucine, aspartate, proline, methionine, etc from proteins and peptides, in order that proteins are properly regulated, targeted for degradation, and trafficked within both animal and plant cells (3). As a result, these enzymes are involved in diverse processes, including meiosis (1), cellular senescence (1), blood pressure control (4, 5), angiogenesis (6), and inflammation (7). PFM18AAP is the sole aspartyl aminopeptidase (AAP) present in the genome of the malaria parasite Plasmodium falciparum (8). It exhibits exopeptidase activity exclusively against the N-terminal acidic amino acids glutamate and aspartate (9-11), is found in all intra-erythrocytic stages of the parasite (9), and functions to complete the hydrolysis of host hemoglobin into amino acids for use in de novo protein synthesis by the parasite (12, 13). Studies demonstrating that genetic knockdown of PFM18AAP results in a lethal parasite phenotype (9), and that inhibitors of methionine (14) and leucine (12, 15) aminopeptidases prevent malaria growth in culture and hemoglobin degradation, suggest that these enzymes are essential for parasite survival. As a result, the identification of selective inhibitors of PFM18AAP would elucidate this enzyme's role in the P. falciparum lifecycle, and serve as potential therapeutic agents to control malaria infection.

The purpose of this assay is to identify compounds that inhibit the activity of M18 aminopeptidase of the malaria parasite Plasmodium falciparum (PFM18AA). In this biochemical assay, a commercially available fluorogenic peptide substrate (H-Glu-NHMec) is incubated with purified recombinant PFM18AAP enzyme (rPFAAP) in the presence of test compounds. Cleavage of the substrate by rPFAAP enzyme liberates the NHMec leaving group from the peptide, leading to increased well fluorescence. As designed, compounds that inhibit PFM18AAP will block rPFAAP-mediated cleavage of H-Glu-NHMec and liberation the NHMec leaving group from the substrate, resulting in decreased well fluorescence as measured at 340 nm excitation and 450 nm emission. Test compounds were assayed in singlicate at a final nominal concentration of 7.35 micromolar.

Protocol Summary:

Prior to the start of the assay, 2.5 microliters of assay buffer (50mM Tris HCl pH7.5, 4mM CoCl2, 0.1% BSA) containing 5micrograms/mL rPFM18AAP were dispensed into a 1536 microtiter plate. Next, 37 nL of test compound in DMSO, ZnCl2 (2mM final concentration), or DMSO alone (0.74% final concentration) were added to the appropriate wells. The plates were then incubated for 30 minutes at 25 degrees Celsius.The assay was started by dispensing 2.5 microliters of 100 micromolar H-Glu-NHMec substrate in buffer (50 mM Tris HCl, pH 8.8) into all wells. Well fluorescence was read immediately (T0) on the Viewlux (Perkin-Elmer) and again after 90 minutes (T90) of incubation at 25 degrees Celsius.Prior to further calculations, T0 was subtracted from T90 for each individual well. The difference between RFU values read at T0 (RFU_T0) and T90 (RFU_T90), named delta RFU, was calculated as follows:delta RFU = RFU_T90 - RFU_T0

The percent inhibition for each well was then calculated as follows:Percent inhibition = (test_compound_delta RFU - negative_control_ delta RFU)/(positive_control_ delta RFU - negative_control_ delta RFU)*100Where:Test_Compound is defined as wells containing test compound.Negative_Control is defined as the median of the wells containing test compounds.Positive_Control is defined as the median of the wells containing ZnCl2.

A mathematical algorithm was used to determine nominally inhibiting compounds in the Primary screen. Two values were calculated: (1) the average percent inhibition of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition than the cutoff parameter was declared active.

Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. In this case the results of each separate campaign were assigned "Active/Inactive" status based upon that campaign's specific compound activity cutoff value. All data reported were normalized on a per-plate basis. In this assay, ZnCl2 had an IC50 of approximately 0.525 uM. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, compounds that modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR.